1. Field of the Invention
The field of the present invention relates generally to the biospecific adhesion of cells to a surface. More specifically, the invention relates to the chemical modification of a surface and the covalent attachment thereto of small peptides to promote cell adhesion. Even more particularly, the surface may comprise a ceramic, a metal or a polymer. The small peptides include a minimal cell receptor recognition amino acid sequence which promotes cell adhesion and is common to a variety of cell adhesion molecules. The surfaces and methods of the present invention thus relate to cell adhesion techniques which are independent of culture media serum composition and adsorbed surface proteins.
2. Description of the Related Art
The interaction of cells with extracellular matrix in vivo is involved in a number of important biological processes, such as the regulation of cellular growth, migration, and differentiation. The role of eukaryotic cell adhesion in culture largely dictates the success of a particular cell culture effort or endeavor. Adhesion, spreading, and contraction on solid substances are prerequisites for growth of normal anchorage dependent cells in vitro (1, 2). This cellular bioadhesion is affected by several factors, including the particular type of cell, the cell culture media used, and the particular surface upon which the cells are cultured.
Many mammalian cells are cultured on polymer surfaces. Nearly all mammalian cell adhesion to synthetic polymer surfaces is controlled by adsorbed proteins and is receptor mediated. Fibronectin (FN), was the first cell adhesion molecule (CAM) that was shown to be involved in the adhesion of some avian and mammalian cell types to extracellular substrates (3, 4). FN is commonly provided to the in vitro environment through the addition of serum, in a form known as cold-insoluble globulin (CIg). For normal attachment and spreading of cells to occur, it was found that CIg had to be adsorbed to the culture surface (5, 6).
FN consists of several protease-resistant domains, each of which contain specific binding sites for other extra cellular molecules and for the cell surface (7). The cell attachment activity has been localized to a tripeptide sequence (RGD), located in the cell binding domain of FN as well as in several other CAMs (8). Substrate-bound, RGD-containing peptide, directly adsorbed to the substrate, or peptide cross-linked to adsorbed albumin or IgG, was found to promote fibroblast attachment and spreading. This attachment and spreading activity was found to be readily inhibited by the addition of soluble RGD-containing peptides to the medium (8).
Affinity chromatography of cellular extracts on cell attachment-promoting FN fragments combined with specific elution utilizing synthetic RGD-containing peptides yielded a receptor with two 140 kD subunits (9). The mammalian FN receptor and other RGD directed receptors are typically heterodimers of two subunits, alpha and beta (10). Families of these receptors consist of members with similar beta subunits, whereas the alpha subunits are more distinct and restrict the receptor's affinity to one or a few CAMs (11). Collectively, these structurally and functionally related receptor families are known as the integrin superfamily (12, 13).
A basic understanding of the molecular mechanisms underlying the process of cell adhesion has thus developed regarding the role of the cell culture substrates and other surfaces in promoting cell adhesion. Basically, after a protein solution is placed on a culture substrate, proteins are immediately adsorbed to the surface. If there are receptors for some of these adsorbed proteins on the cell surface, and if the conformation of the adsorbed protein is not so extensively altered by adsorption as to destroy the high ligand-receptor affinity, then cell adhesion to the culture substrate and cell spreading can result.
If the cells are seeded on a substrate in the absence of adsorbed proteins, then the proteins on the cell surface may directly adsorb to the surface and the cell will, provided favorable conditions, secrete its own proteins toward the surface in the form of an extracellular matrix. However, if the substrate does not support protein adsorption, or if it supports high affinity adsorption of a protein for which there is not a cell-surface receptor, then the substrate will not support cell adhesion. In no case has the cell in culture been found to actually touch the surface except through these intermediate adsorbed proteins.
Some investigators who study short-term cell adhesion have proposed the use of substrate treating systems which promote cell adhesion wherein particular peptides are adsorbed to a polymer surface. For example, Singer, et al. proposed the adsorption of a 13-mer peptide containing the RGD sequence onto a polymer substrate to promote cell adhesion (14). However, peptides of this length have been found to be highly susceptible to degradation at high temperatures and to the proteolytic action of the cultured cells themselves. Additionally, peptides adsorbed to a surface are subject to desorption upon repeated use. Thus, surfaces with long amino acid residue peptides absorbed thereto have been found to be unstable and thus unsuitable in preparing reusable cell culture substrates.
An alternative approach in promoting cell adhesion is through chemical modification of the surface to facilitate the attachment of peptides to the substrate. However, present technology for chemical modification of substrates is particularly non-specific and empirical. For example, treatment of polymer surfaces with various radio frequency plasma discharges, both polymerizing and nonpolymerizing, has been proposed. Alternative approaches of surface acid treatment or surface incorporation of charged groups have also been described. However, these various surface treatments alter only the pattern of protein adsorption on the culture surface, which in turn functions to modify the cells' characteristic adhesion and spreading behavior. Thus, the protein and peptide surface adsorption and desorption problems still remain, limiting the reusability of culture plates and other surfaces so treated.
An alternative to surface adsorption of peptides to promote cell adhesion has been to instead chemically attach peptides to a surface. For example, the method of polymer surface chemical modification was employed by Brandley, et al., (1988) (Analyt. Biochem. 172: 270), who proposed the inclusion of a 9-mer peptide in a polymer substrate to promote cell adhesion (Id.). While enhanced cell adhesion was attained using the Brandley technique, the method required higher concentrations of peptide to promote the same level of cell adhesion observed in the relatively more peptide efficient adsorbed peptide systems. For example, FIG. 1 of Brandley shows surface concentrations of peptide on the average of about 6 nanomoles per square centimeter (Brandley, at pg. 275). These high peptide concentrations suggest the Brandley method does not control for the inclusion of peptide at the polymer surface only, but instead permits the incorporation of peptide throughout the bulk of the polymer. Given that synthetic peptides cost about $5,000 per gram, this method would not facilitate the economical preparation of cell culture substrates commercially.
Thus, a need still exists in the art for an economical system of preparing thermally stable, peptide-coated surfaces with cell adhesion promoting characteristics which are resistant to the desorptive effects of repeated usage and proteolysis by cellular proteases or proteases added to remove cells. A more commercially feasible and economical system would be substantially more peptide-efficient than those proposed by Brandley and others of skill in the art of cell culture and polymer chemistry.
The following abbreviations are used by Applicants throughout the application:
D=Asp (aspartic acid) PA1 F=phenylalanine PA1 G=Gly (glycine) PA1 I=Ile (isoleucine) PA1 R=Arg (arginine) PA1 S=Ser (serine) PA1 Y=Tyr (tyrosine) PA1 CFN=cellular fibronectins PA1 FC=focal contacts PA1 FEP=fluorinated ethylene polymers PA1 fg=fibrinogen PA1 GREDV=glycine, arginine, glutamic acid, aspartic acid, valine or Gly-Arg-Glu-Asp-Val PA1 GRGD=amino acid sequence glycine, arginine, glycine, aspartic acid; or Gly-Arg-Gly-Asp PA1 HFF=human foreskin fibroblast cells PA1 kD=kilodalton PA1 mer=amino acid residue PA1 nm=nanomoles PA1 PAE=porcine aortic endothelial (cells) PA1 PBS=phosphate buffered saline PA1 PDMS=poly(dimethyl siloxane) PA1 PET=poly(ethylene terephthalate) PA1 pFN=plasma fibronectins PA1 PHEMA=poly(hydroxyethyl methacrylate) PA1 PTFE=poly(tetrafluoroethylene) PA1 REDV=arginine, glutamic acid, aspartic acid, valine or Arg-Glu-Asp-Val PA1 RGD=amino acid sequence arginine, glycine, aspartic acid, or Gly-Arg-Gly-Asp PA1 SAM=surface adhesion molecule PA1 ug=micrograms PA1 ul=microliter PA1 YIGSR=tyrosine, isoleucine, glycinem serine, aspartic acid or Try-Ile-Gly-Ser-Arg